Application of peptide chromatography for the isolation of antibodies from bovine skim milk, acid whey and colostrum

Abstract

Protein A mimetic peptide ligands have several benefits over conventional Protein A/G ligands, namely that they are small in size, have low production costs, are stable over a wide range of pH values and can withstand cleaning by harsh sanitization agents such as sodium hydroxide. In this paper, a hexamer peptide (HWRGWV) affinity matrix was used for the isolation of bovine immunoglobulins from various dairy streams (skim milk, acid whey and colostrum). Bound immunoglobulins were recovered in elution buffer (0.2 M sodium acetate buffer, pH 4.0) fractions with a purity of >85% in a single step. The peptide resin has achieved a maximum equilibrium adsorption capacity of 23±0.58 mg.mL-1 of resin for bovine IgG and had a dynamic binding capacity of 11.8±0.03 mg.mL-1 at residence time of 2 min. These results suggest that the hexamer peptide chromatography could potentially be used for the selective purification of bovine immunoglobulins from dairy streams. This method has promise as an alternative to conventional Protein A/G chromatography for direct capture of immunoglobulins from streams containing relatively high immunoglobulin concentrations such as colostrum, transgenic or hyper-immune milk. In the abstract, equilibrium adsorption capacity should appear with the associated error (in the previous submission this error appear).Decision: Authors are accepted the above and reviewed as suggested (23±0.58). Comment2.Page 12 line 10 "However, for experiments with standard bIgG at 1 mg mL-1 and 9 mg mL-1 were overloaded" The idea to answer comment 8, reviewer 1 is there but the sentence should better introduced in the text.Decision: Authors are accepted the above and reviewed as suggested in the draft. "However, for experiments with standard bIgG at 1 mg.mL -1 and 9 mg.mL -1 were overloaded since column reached its dynamic capacity approximately at 5.5 mL for 1 mg.mL -1 bIgG and 1.4 mL for 9 mg.mL -1 bIgG. Due to this, % of IgG bound data presented in table 1.1 for these experiments may not reflect the actual numbers". Thank you.Yours sincerely, Jagan M Billakanti, PhD. Cover LetterRevision 2 Reviewer #1 Comment1. In the abstract, equilibrium adsorption capacity should appear with the associated error (in the previous submission this error appear).Decision: Authors are accepted the above and reviewed as suggested (23±0.58) Comment2. Page 12 line 10 "However, for experiments with standard bIgG at 1 mg mL-1 and 9 mg mL-1 were overloaded" The idea to answer comment 8, reviewer 1 is there but the sentence should better introduced in the text.Decision: Authors are accepted the above and reviewed as suggested in the draft."However, for experiments with standard bIgG at 1 mg.mL -1 and 9 mg.mL -1 were overloaded since column reached its dynamic capacity approximately at 5.5 mL for 1 mg.mL -1 bIgG and 1.4 mL for 9 mg.mL -1 bIgG. Due to this, % of IgG bound data presented in table 1.1 for these experiments may not reflect the actual numbers". Detailed Response to ReviewersHighlights  HWRGWV peptide resin showed an equ...

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

“…Traditional, Ig-binding proteins, generally produced by gram-positive bacteria, have been widely explored in Ab isolation procedures [ 12 , 13 , 14 ]. In particular, Protein A (SpA), a virulence factor produced by Staphylococcus aureus , has been used in the chromatographic purification of Abs due to its high affinity towards IgGs from different species [ 15 ].…”

Criogel monolítico de PAAm-Alg-AGE-IDA

“…Contudo, os métodos cromatográficos ainda são os mais utilizados para a purificação de IgG do soro bovino visando um número menor de etapas, ou até mesmo uma única etapa, e a cromatografia de afinidade mostra-se a mais promissora, mesmo que em escala laboratorial (BILLAKANTI et al, 2014). Em busca de superar obstáculos e tornar o método ainda mais eficiente, simples, com menor custo e possibilitar sua ampliação para larga escala, estudos vêm sendo realizados, como por exemplo, substituindo os ligantes bioespecíficos pelos pseudobioespecíficos (KONECNY et al, 1994;DAOUD-ATTIEH et al, 2013;BILLAKANTI et al, 2014).…”

“…Alguns ligantes pseudobioespecíficos reportados na literatura, como por exemplo os aminoácidos histidina e o imidazol (POURROSTAM-RAVADANAQ et al, 2020), peptídeos (BILLAKANTI et al, 2014) e os íons metálicos imobilizados (FUKUMOTO et al, 1994) tem possibilitado capturar a IgG do soro bovino. Ligantes pseudobioespecíficos apresentam vantagens de possuírem menor tamanho, baixo custo, melhor estabilidade química e física sob condições de alta força iônica e alto e baixo pH (ROQUE et al, 2007;PRASANNA et al, 2015;ARORA et al, 2017).…”